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WO2021030716A1 - Procédé de capture de sonde pour la récupération de vdj de chaîne bêta de tcr alpha et bêta à partir d'arn transcrit inverse d'oligo-dt - Google Patents

Procédé de capture de sonde pour la récupération de vdj de chaîne bêta de tcr alpha et bêta à partir d'arn transcrit inverse d'oligo-dt Download PDF

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Publication number
WO2021030716A1
WO2021030716A1 PCT/US2020/046433 US2020046433W WO2021030716A1 WO 2021030716 A1 WO2021030716 A1 WO 2021030716A1 US 2020046433 W US2020046433 W US 2020046433W WO 2021030716 A1 WO2021030716 A1 WO 2021030716A1
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Prior art keywords
tcr
amplicons
sequences
vdj
beta chain
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PCT/US2020/046433
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Inventor
Wenjing PAN
Miranda BYRNE-STEEL
Brittany BROWN
Li Song
Jian Han
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Irepertoire Inc
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Irepertoire Inc
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Priority to BR112022002805A priority Critical patent/BR112022002805A2/pt
Priority to CA3151004A priority patent/CA3151004A1/fr
Priority to KR1020227008310A priority patent/KR20220076453A/ko
Priority to EP20851793.8A priority patent/EP4013863A4/fr
Priority to US17/635,219 priority patent/US20220220470A1/en
Priority to AU2020328599A priority patent/AU2020328599A1/en
Priority to CN202080063551.6A priority patent/CN114616329A/zh
Publication of WO2021030716A1 publication Critical patent/WO2021030716A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/10—Nucleotidyl transfering
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
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    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/149—Particles, e.g. beads

Definitions

  • TCR T Cell Receptor
  • BCR B Cell Receptor
  • RNA sequencing can provide information related to various genes expressed in the cell traced back to the same cell via the barcode introduced during RT, but this method cannot reliably reveal the sequence of the paired-receptor VDJ-rearrangement.
  • the present disclosure relates to a method comprising the steps of: reverse transcribing at least one first strand of cDNA from mRNA using an oligo-dT primer, wherein the mRNA comprises at least one target sequence to produce a first strand cDNA and wherein the oligo-dT primer comprises an engineered sequence on the 5’ end; producing a first set of amplicons by amplifying the first strand cDNA using a multiplex primer mix, wherein the multiplex primer mix comprises two or more primers configured to bind two or more sequences selected from the group consisting of: TCR alpha chain VDJ and TCR beta chain VDJ sequences, and a reverse primer configured to bind to the engineered sequence on the 5’ end of the oligo-dT primer; using probe beads to capture amplicons comprising TCR alpha chain VDJ sequences and TCR beta chain VDJ sequences from the first set of amplicons, wherein the probe beads comprise primers configured to bind to
  • the method further comprises the step of, after amplifying the eluted amplicons, sequencing the second set of amplicons using next generation sequencing. In some embodiments, the method further comprises the step of, after sequencing the second set of amplicons using next generation sequencing, evaluating the sequences to determine the frequency of TCR alpha chain VDJ sequences and TCR beta chain VDJ sequences.
  • the probe bead primers are configured to bind to at least one location within the TCR alpha chain constant gene region and at least one location within the TCR beta chain constant gene region. In some embodiments of the method, the probe bead primers are configured to bind to at least one location within the TCR alpha chain constant gene region. In some embodiments of the method, the probe bead primers are configured to bind to at least one location within the TCR beta chain constant gene region.
  • the oligo-dT primer comprises a molecular barcode.
  • the multiplex primer mix comprises an engineered sequence on the 5’ end of the primers and which is configured as a universal binding site.
  • a majority of the two or more primers of the multiplex primer mix are configured to favor amplification of the sense strand VDJ sequences. In some embodiments of the method, a majority of the two or more primers of the multiplex primer mix are configured to bind to one or more TCR alpha chain VDJ sequences. In some embodiments of the method, a majority of the two or more primers of the multiplex primer mix are configured to bind to one or more TCR beta chain VDJ sequences.
  • the first set of amplicons is cleaned by SPRI bead selection and an additional round of PCR is performed.
  • FIG. 1 displays four stages of a probe-capture strategy including asymmetrical enrichment of second strand cDNA from targeted multiplex mix product, gene-specific probe hybridization, capture of targeted second-strand cDNA product and removal of off-target products by washing, and two-step PCR and amplification of tagged products.
  • FIG. 2 displays histograms showing next generation sequencing results for TCR alpha chain VDJ, TCR beta chain VDJ, and unrelated to TCR VDJ as a percentage of total reads.
  • A Bulk RNA without probe capture.
  • B Bulk RNA with probe capture.
  • C Single cell without probe capture.
  • D Single cell with probe capture.
  • This disclosure generally relates a method of amplifying TCR alpha chain VDJ and/or
  • TCR beta chain VDJ sequences by reverse transcribing at least one first strand of cDNA from mRNA containing at least one target sequence using an oligo-dT primer, using a multiplex primer mix of primers configured to bind to TCR alpha chain VDJ and/or TCR beta chain VDJ sequences to amplify the cDNA, using probe capture to capture TCR VDJ-specific amplicons and elute non-TCR VDJ-specific amplicons, performing an additional round of PCR to further enrich TCR alpha chain VDJ and TCR beta chain VDJ amplicons, and sequencing the resulting amplicons using next generation sequencing.
  • engineered sequence refers to a nucleotide sequence.
  • An engineered sequence can be configured for a particular purpose.
  • an engineered sequence can be designed to serve as a universal binding site.
  • oligo-dT refers to a homopolymer typically consisting exclusively of thymidines. Preferred length of the oligo-dT molecule is 10 to 100 bases, more preferred is a length of 10 to 30 bases, most preferred is a length of 20 bases.
  • the oligo-dT molecule is preferably blocked at its 3'-hydroxyl group by a blocking group to prevent extension by a polymerase reaction. It is known to those of skill in the art that oligo-dT can also be modified as long as the molecule is capable of hybridizing to polyA-RNA. Modifications are but not limited to all types of thymidine analogs like 2'-deoxyuridine.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • HNA hexitol nucleic acid
  • thymidine or uridine derivatives base modified uracil derivatives like 5-propinyl-uracil, but also modification with labels like fluorescent labels or haptens or modification with nucleotides or nucleotide sequences other than thymidine and uridine.
  • probe bead means a bead to which one or more primers can be adhered and which is suitable for use with one or more purification methods, such as column purification.
  • the term “subject” means a human or other animal.
  • the subject has been immunized or is suffering from an infection, cancer, an autoimmune condition, or any other diseases.
  • the subject can be a human diagnosed with a disease, exhibiting symptoms of a disease, not diagnosed with a disease, or not exhibiting symptoms of a disease.
  • target sequence means a nucleic acid sequence to be detected and which anneals with a probe or primer under hybridization, annealing or amplification conditions.
  • TCR alpha chain and TCR beta chain refer to the alpha and beta chains that comprise the TCR and which assemble to form a heterodimer and associate with the CD3-transducing subunits to form the T-cell receptor complex present on the T cell surface.
  • Each alpha and beta chain of the TCR consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region.
  • V variable
  • C constant
  • the variable region of the alpha and beta chains are generated by V(D)J recombination, creating a large diversity of antigen specificities within the population of T cells.
  • this disclosure relates to a method comprising the steps of: reverse transcribing at least one first strand of cDNA from mRNA, wherein the mRNA comprises at least one TCR alpha chain VDJ or TCR beta chain VDJ sequence, using an oligo-dT primer.
  • the oligo-dT primer comprises an engineered sequence 5’ of the oligo-dT portion.
  • the engineered sequence 5’ of the oligo-dT portion provides the benefit of simplifying downstream amplification.
  • the oligo-dT primer does not comprise an engineered sequence 5’ of the oligo-dT portion.
  • the oligo-dT primer may also comprise a molecular barcode as a unique identifier.
  • a molecular barcode can comprise a nucleotide sequence comprising from about 4 to 15 randomly-generated nucleotides.
  • Molecular barcodes can be used as disclosed in US20150132754 (Wang, et al.) and US20120171725 (Han), which are incorporated herein by reference.
  • the mRNA can be isolated from any source comprising one or more T cells.
  • the mRNA can be isolated from peripheral blood or other biological samples from a subject. Methods of extracting mRNA are known to those of skill in the art.
  • the first strand cDNA produced after reverse transcription with the oligo-dT primer is then amplified using a targeted multiplex mix of primers covering either TCR alpha chain VDJ only or TCR beta chain VDJ only, or both TCR alpha chain and beta chain VDJ.
  • the VDJ- primer mixes may also contain an engineered sequence on the 5’ end to provide a universal binding site for downstream PCR.
  • the multiplex PCR mix comprises an optimized primer set of VDJ sequences of interest and a reverse primer for the engineered sequence located on the 5’ end of the oligo-dT primer during reverse transcription ( Figure 1).
  • PCR is performed using an asymmetric balance of primers configured to favor production of the sense strand VDJ.
  • the PCR product of the first amplification is cleaned by either SPRI bead selection or another standard PCR clean-up method, and the PCR is repeated to increase the template amount.
  • probe beads are generated using constant region capture primers specific for the TCR alpha chain constant gene region and the TCR beta chain constant gene region. These capture sequences can be for one specific location or multiple locations within the junction or constant gene for their respective chains. The location of the junction or constant probe primer is also an important consideration as some positions are better than others for VDJ sequence recovery.
  • unbound species are washed away - for example by using column purification methods known to those of skill in the art - and the captured sequences are eluted from the beads.
  • Another round of PCR is then performed to both enrich the template again and assign additional indices for sequencing.
  • the library is then sequenced using next generation sequencing, and the data are evaluated for frequency of TCR alpha chain and beta chain VDJ sequences.
  • VDJ information for the alpha chain was consistently very difficult to capture from an oligo- dT reverse transcribed cDNA, regardless of whether the system was a single cell capture system or a bulk RNA reverse-transcribed by an oligo-dT primer. Since this information forms one of the chains of receptor pair, it is critical that this information also be recovered.
  • An advantage of the disclosed methods is to consistently recover both the TCR alpha and beta chain VDJ-rearrangement information using a targeted-sequencing approach coupled to probe-capture. These methods rescue the sequence of the TCR alpha chain VDJ, while also increasing the number of VDJ-specific sequencing reads.
  • the presently disclosed methods result in a much higher capture of alpha chain sequences relative to conventional methods, thereby increasing the discovery of the alpha chain immune repertoire and the overall receptor pairing success rate.
  • the methods also enable an increased discovery of TCR beta chain sequences.
  • the reduction in the number of off-target sequencing reads offered by the disclosed methods results in overall reduced experiment associated costs, because less sequencing depth is required to maximize the clonotype discovery for each chain. As a result, both immune repertoire discovery and single cell pairing from an oligo-dT reverse transcribed template is improved by the described methods.
  • TCR alpha chain VDJ The TCR alpha chain VDJ, the TCR beta chain VDJ, and both together were amplified on reverse transcribed cDNA on bulk RNA. Analysis of the NGS data revealed approximately 90-94% of the data was unrelated to the TCR VDJ sequences, approximately 8% of TCR beta chain sequences and approximately 6% of TCR alpha chain sequences. When the TCR alpha and beta chain sequences were co-amplified, approximately 92% of the data were unrelated to the VDJ sequences, with 5.8% TCR beta chain sequences, and 1.9% TCR alpha chain sequences.
  • the off-target sequencing results were re-analyzed for the percentages of various components to see if certain V-alpha sequences could be modified or removed to increase the percentage of on-target V-alpha results. Using all of the information related to the off- target amplicons, the entire primer system was redesigned to avoid off-target results. The analysis on applicant’s bulk RNA oligo-dT system was repeated. Analysis of the NGS data reveals 90% of the data were unrelated to the TCR VDJ sequences (trash) and 6-7% of TCR alpha chain sequences.
  • NGS data reveals by using no probe pull down for TCR alpha chain only, 85% of the data were unrelated to the TCR VDJ (trash). TCR beta chain only reveals 87% of the data were unrelated to the TCR VDJ.
  • TCR alpha chain sequences When co-amplifying both TCR alpha and beta chain sequences using no probe to capture, 73% of the data were unrelated to the TCR VDJ sequences, 24% of TCR beta chain sequences and 2% of TCR alpha chain sequences ( Figure 2-A).
  • Applicants used the probe-capture system after asymmetrical enrichment of second strand cDNA products. The increase in usable TCR alpha and beta chain data with respect to non-probe capture is 7-fold.
  • TCR alpha and beta chain data with respect to non-probe capture is 7-fold.
  • 25% of TCR alpha chain data were unrelated to the TCR VDJ sequences and 29% of TCR beta chain data were unrelated.
  • 19% of the data were unrelated to the TCR VDJ sequences, 51 % of TCR beta chain sequences and 28% of TCR alpha chain sequences ( Figure 2-D).

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Abstract

La présente invention concerne de manière générale un procédé d'amplification de séquences VDJ à chaîne alpha de TCR et/ou de chaîne bêta de TCR par transcription inverse d'au moins un premier brin d'ADNc à partir d'ARNm contenant au moins une séquence cible à l'aide d'une amorce oligo-dT, à l'aide d'un mélange d'amorces multiplex d'amorces configurées pour se lier à des séquences VDJ de chaîne alpha de TCR et/ou de chaîne bêta de TCR pour amplifier l'ADNc, à l'aide d'une capture de sonde pour capturer des amplicons spécifiques de VDJ de TCR et éluer des amplicons spécifiques de VDJ non TCR, la réalisation d'un cycle supplémentaire de PCR pour enrichir davantage les amplicons VDJ de chaîne alpha de TCR et de chaîne bêta de TCR, et le séquençage des amplicons résultants à l'aide d'un séquençage de nouvelle génération.
PCT/US2020/046433 2019-08-14 2020-08-14 Procédé de capture de sonde pour la récupération de vdj de chaîne bêta de tcr alpha et bêta à partir d'arn transcrit inverse d'oligo-dt Ceased WO2021030716A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
BR112022002805A BR112022002805A2 (pt) 2019-08-14 2020-08-14 Método de captura por sonda para recuperação de vdj da cadeia alfa e beta de tcr por rna transcrito de forma reversa baseado em óligo-dt
CA3151004A CA3151004A1 (fr) 2019-08-14 2020-08-14 Procede de capture de sonde pour la recuperation de vdj de chaine beta de tcr alpha et beta a partir d'arn transcrit inverse d'oligo-dt
KR1020227008310A KR20220076453A (ko) 2019-08-14 2020-08-14 올리고-dT 역전사된 RNA로부터 TCR 알파 및 베타 쇄 VDJ를 회수하는 프로브 포획 방법
EP20851793.8A EP4013863A4 (fr) 2019-08-14 2020-08-14 Procédé de capture de sonde pour la récupération de vdj de chaîne bêta de tcr alpha et bêta à partir d'arn transcrit inverse d'oligo-dt
US17/635,219 US20220220470A1 (en) 2019-08-14 2020-08-14 Probe-capture method for tcr alpha and beta chain vdj-recovery from oligo-dt reverse transcribed rna
AU2020328599A AU2020328599A1 (en) 2019-08-14 2020-08-14 Probe-capture method for TCR alpha and beta chain VDJ-recovery from oligo-dT reverse transcribed RNA
CN202080063551.6A CN114616329A (zh) 2019-08-14 2020-08-14 从OLIGO-DT逆转录RNA中回收TCRα和β链VDJ的探针捕获方法

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US62/886,663 2019-08-14

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EP (1) EP4013863A4 (fr)
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US12112833B2 (en) 2020-02-04 2024-10-08 10X Genomics, Inc. Systems and methods for index hopping filtering
US12168801B1 (en) * 2020-07-02 2024-12-17 10X Genomics, Inc. Hybrid/capture probe designs for full-length cDNA
CN119932009A (zh) * 2025-04-07 2025-05-06 青岛百创智能制造技术有限公司 一种时空转录组特异富集的方法及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120171725A1 (en) 2008-04-03 2012-07-05 Jian Han Amplicon Rescue Multiplex Polymerase Chain Reaction for Amplification of Multiple Targets
US20150132754A1 (en) 2012-05-14 2015-05-14 Cb Biotechnologies, Inc. Method for increasing accuracy in quantitative detection of polynucleotides
US20150329891A1 (en) 2013-12-30 2015-11-19 Atreca, Inc. Analysis of nucleic acids associated with single cells using nucleic acid barcodes
US9512487B2 (en) 2008-11-07 2016-12-06 Adaptive Biotechnologies Corp. Monitoring health and disease status using clonotype profiles
WO2018013723A1 (fr) * 2016-07-14 2018-01-18 Fluidigm Corporation Séquençage de produits de transcription unicellulaires
WO2018075693A1 (fr) 2016-10-19 2018-04-26 10X Genomics, Inc. Procédés et systèmes de codage de molécules d'acide nucléique provenant de cellules individuelles ou de populations de cellules
US20180208984A1 (en) 2017-01-17 2018-07-26 Life Technologies Corporation Compositions and methods for immune repertoire sequencing

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104263818B (zh) * 2014-09-02 2016-06-01 武汉凯吉盈科技有限公司 基于高通量测序技术的全血免疫组库探测方法
CN107208157B (zh) * 2015-02-27 2022-04-05 贝克顿迪金森公司 用于条形编码核酸以用于测序的方法和组合物

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120171725A1 (en) 2008-04-03 2012-07-05 Jian Han Amplicon Rescue Multiplex Polymerase Chain Reaction for Amplification of Multiple Targets
US9512487B2 (en) 2008-11-07 2016-12-06 Adaptive Biotechnologies Corp. Monitoring health and disease status using clonotype profiles
US20150132754A1 (en) 2012-05-14 2015-05-14 Cb Biotechnologies, Inc. Method for increasing accuracy in quantitative detection of polynucleotides
US20150329891A1 (en) 2013-12-30 2015-11-19 Atreca, Inc. Analysis of nucleic acids associated with single cells using nucleic acid barcodes
WO2018013723A1 (fr) * 2016-07-14 2018-01-18 Fluidigm Corporation Séquençage de produits de transcription unicellulaires
WO2018075693A1 (fr) 2016-10-19 2018-04-26 10X Genomics, Inc. Procédés et systèmes de codage de molécules d'acide nucléique provenant de cellules individuelles ou de populations de cellules
US20180208984A1 (en) 2017-01-17 2018-07-26 Life Technologies Corporation Compositions and methods for immune repertoire sequencing

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HANSON ET AL., JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 138, no. 35, 2016, pages 11073 - 11076
See also references of EP4013863A4

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EP4013863A1 (fr) 2022-06-22
EP4013863A4 (fr) 2023-08-30
CN114616329A (zh) 2022-06-10
CA3151004A1 (fr) 2021-02-18
KR20220076453A (ko) 2022-06-08
BR112022002805A2 (pt) 2022-08-09
AU2020328599A1 (en) 2022-03-31

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